1. Field of the Invention
The present invention relates to enzyme electrodes for measuring analyte concentration in fluids, for example glucose in whole blood. Enzyme electrodes comprise an enzyme layered on or mixed with an electrically conductive substrate. The electrodes respond amperometrically to the catalytic activity of the enzyme in the presence of a suitable analyte (substrate). The invention also extends to a biosensor, notably a single-use biosensor, which includes the enzyme electrode.
2. Description of the Prior Art
Amperometric biosensors are well known in the art. Typically the enzyme is an oxidoreductase, for example glucose oxidase, cholesterol oxidase, or lactate oxidase, which produces hydrogen peroxide according to the reaction:analyte+O2−[oxidase]→oxidised product+H2O2.
The peroxide is oxidised at a fixed-potential electrode as follows:H2O2→O2+2H++2e−.
Electrochemical oxidation of hydrogen peroxide at the platinum centres on the electrode results in transfer of electrons from the peroxide to the electrode producing a current which is proportional to the analyte concentration. Where glucose is the analyte, the oxidised product is gluconolactone. Japanese Unexamined Patent Publication No. 56-163447 describes a system which employs glucose oxidase immobilised on a platinum electrode. The electrode comprises a layer of immobilised enzyme on an electrically conductive carbon base. The base is formed from moulded graphite containing up to 10 parts by weight of a fluorocarbon resin binder, onto which is deposited a thin (less than 1 μm) platinum film. The invention is said to avoid the problems associated with the immobilisation of the enzyme directly onto the platinum surface and to produce an enzyme electrode having rapid response times (5 seconds), high sensitivity and durability. However, according to U.S. Pat. No. 4,970,145, recent experimental work with such electrodes has failed to elicit such benefits.
U.S. Pat. No. 4,970,145 describes an enzyme electrode comprising a substantially heterogeneous porous substrate consisting essentially of resin-bonded carbon or graphite particles with a platinum-group metal dispersed substantially uniformly throughout the substrate, and a catalytically active quantity of an enzyme adsorbed or immobilised onto the surfaces of the porous substrate. The electrodes are manufactured either by cross-linking the enzyme to the substrate, or by suspending the porous substrate in a buffered solution of the enzyme for 90 minutes at room temperature. Alternatively, adsorption of the enzyme to the electrode is effected by electroadsorption, wherein the electrode base material is suspended at a positive potential in an enzyme solution for 60 minutes. The electrode is said to have fast response times (1-2 seconds without a protective membrane, and 10 to 30 seconds with a membrane) and good stability. The working range is said to be extended, and the electrode requires a substantially lower operating potential than normal (325 mV against the more usual 650 mV) and exhibits low background at the operating potential.
U.S. Pat. No. 5,160,418 discloses a simplified enzyme electrode comprising a thin film of a substantially homogeneous blend of enzyme and finely-divided platinum group metal or oxide. Optionally, platinised or palladised finely-divided carbon or graphite may be used and, also optionally, a binder. The film can be made by screen-printing a liquid suspension containing the components.
We have found that prior art systems such as described above have high intercepts relative to sensitivity, resulting in poor calibrated precision. We have also found that there is a gradual attenuation of sensitivity with time which is not necessarily related to enzyme instability.
As an alternative to measurement of an electrical signal following transfer of electrons from peroxide to the electrode, some biosensors include an electron carrier, or “mediator” which, in an oxidised form, accepts electrons from the enzyme and then, in a reduced state, transports the electrons to the electrode where it becomes re-oxidised. Prior art examples of mediators include ferrocene, ferrocene derivatives, ferricyanide, osmium complexes, 2,6-dichlorophenolindophenol, Nile Blue, and Medola Blue; see, for example: U.S. Pat. Nos. 5,708,247, 6,241,862, WO 98/55856, and WO 99/13100. Biosensors that employ a redox mediator to transfer electrons between the enzyme and the electrode will be referred to as “mediated biosensors”.
Mediated biosensors can suffer from a number of problems, including chemical instability. The mediator must be in a particular redox state to function, so that if the reduced form is oxidised by air the measured current will be reduced. Oxygen may also interfere by accepting electrons to form peroxides which are not oxidised at the potential of the mediated electrode. If the electrode potential is increased to oxidise the peroxide, this makes the system prone to interference from other species which may be dissolved in blood, for example paracetamol, ascorbate, and uric acid. Thus, variation in oxygen concentration in blood may cause variation in measured glucose response in a mediated system.
Desirable attributes for a single use biosensor include:                low intercept, related to background—to achieve low coefficients of variation (CV's) after calibration;        as high a sensitivity as the electronics will allow;        stability;        good precision;        reproducible manufacture;        rapid response;        low cost.        
The present invention seeks to provide an enzyme electrode and biosensor which are improved in respect of at least some of the above criteria.